Acyl-coenzyme A:cholesterol acyltransferase (ACAT) converts free cholesterol to cholesterol ester, and is one of the key enzymes in cellular cholesterol metabolism. The first ACAT gene (Acat1, also named Soat1) was identified in 1993 (Chang et al. 1993. J. Biol. Chem. 268:20747-20755). The ACAT enzyme family includes ACAT1, ACAT2 (Oelkers et al. 1998. J. Biol. Chem. 273:26765-26771), and acyl-coenzyme A:diacylglycerol acyltransferase 1 (DGAT1) (Buhman et al. 2000. Biochim. Biophys. Acta 1529:142-154). These enzymes are founding members of the membrane-bound O-acyltransferase enzyme family (MBOAT). MBOATs are multi-span membrane proteins that utilize long-chain or medium-chain fatty acyl-coenzyme A and a hydrophobic substance as their substrates (Hofmann, K. 2000. Trends Biochem. Sci. 25:111-112). Additional MBOATs include ghrelin octanoyl-coenzyme A acyltransferase (Yang et al. 2008. Cell 132:387-396) and lysophospholipid acyltransferases (LPATs; Shindou, H. and T. Shimizu. 2009. J. Biol. Chem. 284:1-5). See also, Chang C. Y. et al. 2011. Front. Biol. DOI 10.1007/s11515-011-1149-z.
Research on the therapeutic utility of ACAT1 and ACAT2 has focused on these enzymes as potential drug targets for treating dyslipidemia and atherosclerosis (Chang et al. 2009. Am. J. Physiol. 297:E1-E9). In addition, recent work has identified ACAT1 as a potential therapeutic target for treatment of Alzheimer's disease (AD) (Bryleva et al. 2010. Proc. Natl. Acad. Sci. 107:3081-3086).
Whether ACAT inhibitors may serve as effective anti-atherosclerosis drugs is under debate (Leon et al. 2005. Pharm. Res. 22:1578-1588; Feng et al. 2003. Nat. Cell Biol. 5:781-792; Nissen et al. 2006. NEJM 354:1253-1263; Rudel, L. L. and R. V. J. Farese. 2006. NEJM 354:2616-2617; Chang et al. 2006. Acta Biochim. Biophys. Sin. 38:151-156; Terasaka et al. 2007. Atherosclerosis 190:239-247; Meuwese et al. 2009. JAMA 301:1131-1139; Parini et al. 2009. JAMA 302:255; Dimmitt, S. and G. Watts. 2009. JAMA 302:255-256). Almost all of the ACAT inhibitors available to date were designed based on initial research from the 1980's and 1990's. The compounds were identified by using conventional biochemical assays based on the ability to inhibit ACAT activity in vitro. In vitro assays, for example, monitor levels of enzyme activity by measuring formation of radiolabeled cholesteryl oleate in microsomal fractions of mammalian cells (e.g., Erickson et al. 1980 J. Lipid Res. 930-941).
In studies using animal models for AD, the ACAT inhibitor CP113818 was shown to inhibit the processing of both human amyloid precursor protein (APP) and mouse APP; a different ACAT inhibitor CI1011 decreased the mature/immature ratio of human APP (Huttune et al. 2009. FASEB J 23:3819-3828). In contrast, Bryleva et al. (2010. Proc. Natl. Acad. Sci. 107:3081-3086) showed that in a similar mouse model for AD, ACAT1 gene knock out (Acat1−/−) was associated with a decrease in full-length human APP protein content; however, there was no effect on mouse APP at any level, and no alteration in the ratio of mature human APP/immature human APP. In contrast to these effects in knockout mice (Acat1−/−), CP113818 did not cause a reduction in full-length human APP content (Hutter-Paier et al. 2004. Neuron 44:227-238). The apparent discrepancy in results raises questions about the specificity of the ACAT inhibitors employed by various investigators. Although CP113818, CI1011 and other structurally related compounds are designated as ACAT inhibitors, they may also inhibit other enzymes in the MBOAT family. For instance, when fed to animals, CI1011 reduces the VLDL-triglyceride content, presumably due to its ability to inhibit DGAT1 in addition to inhibiting ACAT (Llayerias et al. 2003. Cardiovasc. Drug Rev. 21:33-50). Another ACAT inhibitor, CI976, widely used to block cholesteryl ester formation in experimental atherosclerosis research (Bocan et al. 1991. Arterioscler. Thromb. 11:1830-1843), has been shown to inhibit a unique, lysohpospholipid acyltransferase (LPAT; Brown et al. 2008. Traffic 9:786-797).
An additional concern regarding currently available ACAT inhibitors is that many ACAT inhibitors are hydrophobic, membrane active molecules (Homan, R. and K. L. Hamelehle 2001. J. Pharm. Sci. 90:1859-1867). The hydrophobicity of the ACAT inhibitors may enable these compounds to have easier access to the active sites of ACAT/MBOAT, which are often buried within the membrane bilayer (Chang et al. 2009. Am. J. Physiol. 297:E1-E9). An undesirable property of these membrane-active compounds is that in intact cells, these compounds may partition into membranes at relatively high concentration and perturb membrane properties in a nonspecific manner. Thus, the currently available ACAT inhibitors may cause membrane deformations in neurons and in other cell types. Novel ACAT1 and ACAT2 inhibitors are sought that have increased specificity and novel binding properties.